C. trachomatis is the most common reportable sexually transmitted infection (STI) and is responsible for > 1 million cases in the U.S. and approximately 92 million cases worldwide each year. Genital infection can lead to immune-mediated damage of the female reproductive organs and serious reproductive disability, including pelvic inflammatory disease (PID) that can result in chronic pelvic pain, ectopic pregnancy and infertility. Approximately 8% of females annually develop PID and this risk significantly increases by 40-70% following re- infection. The reinfection rate is approximately 13% and occurs within 6 months. Viewed in economic terms, treatment of chlamydial STIs and treatment of PID costs the US health care system billions of dollars annually. This project examines a potential contributing factor for developing PID/infertility and the results can be used for development of therapeutics to prevent PID/infertility and ultimately reduce treatment costs. The mouse model of C. trachomatis genital infection (C. muridarum) is used to reveal the underlying mechanism(s) for developing PID/tubal infertility and is called upper genital tract pathology (UGTP). Our in vitro and in vivo studies show that iNKT are activated by infection with C. muridarum as others have confirmed. It is well known that iNKT cells modulate the outcome of immune responses and likely play a role in the immune response against Chlamydia. In order to understand how iNKT cells modulate anti-chlamydial immune, ie aid in eradiation or contribute to female reproductive tract dysfunction, the identity of CD1d-binding glycolipids are needed. Our previous data revealed that glycolipid antigens contained within chlamydiae activate type I (iNKT) and type II NKT cells in vitro using a cell-free CD1d antigen presentation assay. A type II microbial antigen has not yet been identified for any microbe making Chlamydia unique for the study of NKT cell biology. Lipids are important for survival of the obligate intracellular chlamydiae which synthesize a few lipids and incorporate others into their cell wall from host cells. It is not surprising that CD1d degradation is a strategy for immune evasion of these bacteria. Specific Aim 1, we propose to identify CD1d-binding antigens contained within chlamydiae that activate NKT cells in collaboration with Dr. William Hildebrand, an expert in isolating CD1d- binding glycolipids. In Specific Aim 2, we will validate which glycolipids activate iNKT cells in vivo and demonstrate the extent to which activated iNKT cells modulate the outcome of chlamydial genital infection by measuring bacterial burden, Th1 cell numbers, cytokine and chemokine secretion and UGTP in vivo using flow cytometry and ELISA assays on homogenized tissue from mice that lack iNKT cells (CD1d-/- on C57BL/6 Bkgd) after chlamydial genital infection. Understanding how iNKT cells influence chlamydial genital infection is an important question of NKT cell biology and will be studied in collaboration with Dr. Mitchell Kronenberg, an expert in NKT cell biology.